GTPase activating effect of ARHGAP25 is essential for its negative regulatory role in phagocytosis. (A-B) Western blot analysis of whole cell lysate of COSphoxFcγR cells transfected with control vector, cyan fluorescent protein (CFP)–tagged ARHGAP25R192A, CFP-ARHGAP25, or CFP-p50RhoGAP. (C-J) Opsonized yeast uptake by COSphoxFcγR cells. Phagocytosis of cells transfected with control vector (blue; C,G), CFP-ARHGAP25R192A (D,H), or CFP-p50RhoGAP (F,J) is similar to nontransfected cells (E). Cells expressing CFP-ARHGAP25 (blue) are able to bind yeast particles (red); however, the engulfment is inhibited (E,I). P40^Phox is shown in green. (K-L) Quantification of phagocytosis by COSphoxFcγR cells. Panel K shows the number of cells that had phagocytosed 2 or more yeast particles from hundreds of cells transfected with the indicated constructs (phagocytosis %). Panel L shows the number of yeast particles phagocytosed per 100 COSphoxFcγR cells (phagocytosis index). Mean + SEM of 3 separate experiments is shown (*P < .05 compared with control).